Compositions for an orally active 1,2,4-oxadiazole for the treatment of disease

ABSTRACT

The present invention relates to specific doses of and dosing regimens for using a 1,2,4-oxadiazole benzoic acid compound in treating or preventing diseases associated with nonsense mutations. In particular, the invention relates to specific doses and dosing regimens for the use of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid in mammals having diseases associated with nonsense mutations.

This application is a continuation of U.S. application Ser. No.11/918,114, filed Oct. 11, 2008, currently allowed, which is a U.S.national stage of PCT/US2006/012887, filed Apr. 6, 2006, which claimsthe benefit of U.S. provisional application No. 60/787,395, filed Mar.30, 2006 and which claims the benefit of U.S. provisional applicationNo. 60/669,789, filed Apr. 8, 2005, each of which is incorporated byreference herein in its entirety.

1. FIELD OF THE INVENTION

The present invention relates to specific doses of, and dosing regimensfor, using a 1,2,4-oxadiazole benzoic acid compound in treating orpreventing diseases associated with nonsense mutations. In particular,the invention relates to specific doses and dosing regimens for the useof 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid in mammalshaving diseases associated with nonsense mutations.

2. BACKGROUND OF THE INVENTION

A new class of 1,2,4-oxadiazole compounds and their use to treat,prevent or manage diseases ameliorated by modulation of prematuretranslation termination or nonsense-mediated mRNA decay is described inU.S. Pat. No. 6,992,096 B1, issued Jan. 31, 2006, entitled“1,2,4-Oxadiazole Benzoic Acid Compounds and Their Use For NonsenseSuppression and the Treatment of Disease,” which is incorporated hereinby reference in its entirety. One such compound is3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid. As with alldrugs, proper doses and dosing regimens for treating patients havingdiseases such as Cystic Fibrosis and Duchenne Muscular Dystrophy areessential for achieving a desired or optimal therapeutic effect withoutadverse or unwanted effects.

Therefore, a need exists for safe, effective, and non-toxic doses anddosing regimens that either prevent or reduce any adverse or unwantedeffects or provide an optimal therapeutic effect or both, that is,provide a desirable therapeutic profile.

3. SUMMARY OF THE INVENTION

The invention encompasses dosing regimens wherein specific doses of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof areadministered at specific time intervals to modulate prematuretranslation termination or nonsense-mediated mRNA decay, or ameliorateone or more symptoms associated therewith, while reducing or avoidingadverse effects or unwanted effects. The invention further encompassesspecific doses and unit dosage forms of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof.

In one embodiment, the invention relates to methods for administering toa patient in need thereof an effective amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof one, two orthree times in the course of a 24 hour period. The invention alsorelates to methods for administering to a patient in need thereof apharmaceutical composition comprising an effective amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof one, two orthree times in the course of a 24 hour period. The dose at eachadministration during a 24 hour period can be the same or different. Inone embodiment, when administered three times in a 24 hour period, thedose at the first two administrations is the same and the third dose istwice the first dose. In another embodiment, all three doses are thesame.

In another embodiment, the invention relates to methods for treating,preventing or managing a disease ameliorated by modulation of prematuretranslation termination or nonsense-mediated mRNA decay comprisingadministering to a patient in need thereof an effective amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof one, two orthree times in the course of a 24 hour period. Preferably, theadministration is made three times per day continuously, or with a restperiod, for a number of days, weeks, months or years.

The invention also relates to methods for treating, preventing ormanaging a disease ameliorated by modulation of premature translationtermination or nonsense-mediated mRNA decay comprising administering toa patient in need thereof a pharmaceutical composition comprising aneffective amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof one, two orthree times in the course of a 24 hour period. Preferably, theadministration is made three times per day continuously, or with a restperiod, for a number of days, weeks, months or years.

In one embodiment, the invention relates to a method of administering3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof wherein theactive agent is administered to a patient in need thereof one, two orthree times in a 24 hour period, wherein each administration ispreferably separated by about 4-14 hours. In a particular embodiment,the dose of active agent is escalated from the first to third dose.

In another embodiment, the invention relates to continuous therapywherein 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof isadministered to a patient in need thereof for a certain period of time(e.g., 5, 7, 10, 14, 20, 24, 28, 60 or 120 days or longer).

In another embodiment, the invention relates to the administration of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof in singleor divided (e.g., three times daily) doses between 0.1 mg/kg and 500mg/kg, 1 mg/kg and 250 mg/kg, 1 mg/kg and 150 mg/kg, 1 mg/kg and 100mg/kg, 1 mg/kg and 50 mg/kg, 1 mg/kg and 25 mg/kg, 1 mg/kg and 10 mg/kgor 2 mg/kg and 10 mg/kg to a patent in need thereof. In a particularembodiment, 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acidor a pharmaceutically acceptable salt, solvate or hydrate thereof isadministered in a dose of about 4 mg/kg, about 7 mg/kg, about 8 mg/kg,about 10 mg/kg, about 14 mg/kg or about 20 mg/kg. In another embodiment,any dose of the 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid or a pharmaceutically acceptable salt, solvate or hydrate describedin the preceding embodiment is administered one, two or three times in a24 hour period.

In another embodiment, the invention relates to unit dosage formulationsthat comprise between about 35 mg and about 1400 mg, about 125 mg andabout 1000 mg, about 250 mg and about 1000 mg, or about 500 mg and about1000 mg of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid ora pharmaceutically acceptable salt, solvate or hydrate thereof.

In another embodiment, the invention relates to unit dosage formulationsthat comprise 35 mg, 50 mg, 70 mg, 100 mg, 125 mg, 140 mg, 175 mg, 200mg, 250 mg, 280 mg, 350 mg, 500 mg, 560 mg, 700 mg, 750 mg, 1000 mg or1400 mg of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid ora pharmaceutically acceptable salt, solvate or hydrate thereof. In apreferred embodiment, the invention relates to unit dosage formulationsthat comprise 125 mg, 250 mg or 1000 mg of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof.

In another embodiment, the invention relates to a method of maintaininga plasma concentration of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof of greaterthan about 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, about 5 μg/ml, about10 μg/ml, about 20 μg/ml, about 25 μg/ml, about 40 μg/ml, about 50μg/ml, 100 μg/ml, 200 μg/ml, 300 μg/ml, 400 μg/ml, or 500 μg/ml in apatient for at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 12or 24 hours or longer, comprising administering an effective amount3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof to apatient in need thereof

4. DETAILED DESCRIPTION 4.1 Definitions

As used herein, “premature translation termination” refers to the resultof a mutation that changes a codon corresponding to an amino acid to astop codon.

As used herein, “nonsense-mediated mRNA decay” refers to any mechanismthat mediates the decay of mRNAs containing a premature translationtermination codon. In a particular embodiment, the nonsense-mediatedmRNA decay results from a nonsense mutation of DNA.

As used herein, a “premature termination codon” or “premature stopcodon” refers to the occurrence of a stop codon where a codoncorresponding to an amino acid should be.

As used herein, a “nonsense mutation” is a point mutation changing acodon corresponding to an amino acid to a stop codon. In a particularembodiment, the nonsense mutation is a mutation that occurs in DNA andis then transcribed into mRNA.

As used herein, “nonsense suppression” refers to the inhibition orsuppression of premature translation termination and/ornonsense-mediated mRNA decay. In a particular embodiment, the mRNA decayresults from a nonsense mutation of DNA.

As used herein, “modulation of premature translation termination and/ornonsense-mediated mRNA decay” refers to the regulation of geneexpression by altering the level of nonsense suppression. For example,if it is desirable to increase production of a defective protein encodedby a gene with a premature stop codon, i.e., to permit readthrough ofthe premature stop codon of the disease gene so translation of the genecan occur, then modulation of premature translation termination and/ornonsense-mediated mRNA decay entails up-regulation of nonsensesuppression.

As used herein, the terms “adverse effect(s)” or “side effect(s)”include, but are not limited to, nausea, vomiting, diarrhea, headache,elevated serum alanine aminotransferase (ALT), elevated serum aspartateaminotransferase (AST), dizziness, elevated serum creatine kinase (CK),abdominal pain, abdominal gas, eye pain, eye swelling, eye burning,nipple sensitivity, breast tenderness, musculoskeletal chest pain, rash,itching, painful submaxillary lymph node, elevated serum lactatedehydrogenase (LDH), elevated serum aldolase and elevated serumtriglycerides.

As used herein, the terms “active agent,” “drug,” and “drug substance”refer to 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof.

As used herein, the term “dose(s)” means a quantity of active agent tobe administered at one time.

As used herein, the term “unit dosage form(s)” includes tablets;caplets; capsules, such as soft elastic gelatin capsules; sachets;cachets; troches; lozenges; dispersions; powders; solutions; gels;liquid dosage forms suitable for oral or mucosal administration to apatient, including suspensions (e.g., aqueous or non-aqueous liquidsuspensions), emulsions (e.g., oil-in-water emulsions, or a water-in-oilliquid emulsion), solutions, and elixirs; and sterile solids (e.g.,crystalline or amorphous solids) that can be reconstituted to provideliquid dosage forms suitable for oral or parenteral administration to apatient. The unit dosage form does not necessarily have to beadministered as a single dose.

As used herein, the terms “dosing regimen” and “dosage(s)” mean theamount of active agent given per time unit and the duration ofadministration.

As used herein, the term “patient” means an animal (e.g., cow, horse,sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, guineapig, etc.), preferably a mammal such as a non-primate and a primate(e.g., monkey and human), most preferably a human. In certainembodiments, the patient is a fetus, embryo, infant, child, adolescentor adult. In one embodiment, it has been determined throughpre-screening that the patient possesses a nonsense mutation. In anotherembodiment, it has been determined through pre-screening which nonsensemutation the patient has (i.e., UAA, UGA, or UAG).

As used herein, an “effective amount” refers to that amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof sufficientto provide a therapeutic benefit in the treatment or management of thedisease or to delay or minimize symptoms associated with the disease. Inone embodiment, the term “effective amount” refers to the amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof sufficientto achieve a desired plasma level for a certain duration of time.Preferred effective amounts are specifically described herein.

As used herein, the terms “manage”, “managing” and “management” refer tothe beneficial effects that a patient derives from the administration of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof, which doesnot result in a cure of the disease.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the onset, recurrence, spread or worsening of thedisease or a symptom thereof in a patient resulting from theadministration of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid or a pharmaceutically acceptable salt, solvate or hydrate thereof.Because diseases associated with a nonsense mutation can be genetic, apatient can be screened for the presence of a nonsense mutation. In thecase where it is determined through screening that a patient has anonsense mutation, an effective amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof can beadministered to the patient to prevent the onset, recurrence, spread orworsening of the disease or a symptom thereof.

As used herein, the terms “treat”, “treating” and “treatment” refer tothe eradication or amelioration of the disease or symptoms associatedwith the disease. In certain embodiments, such terms refer to minimizingthe spread or worsening of the disease resulting from the administrationof 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof to apatient with such a disease.

As used herein, the term “pharmaceutically acceptable salts” refer tosalts prepared from pharmaceutically acceptable non-toxic acids or basesincluding inorganic acids and bases and organic acids and bases.Suitable pharmaceutically acceptable base addition salts for3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid include, butare not limited to, metallic salts made from aluminum, calcium, lithium,magnesium, potassium, sodium and zinc or organic salts made from lysine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitablenon-toxic acids include, but are not limited to, inorganic and organicacids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic,galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic,phosphoric, propionic, salicylic, stearic, succinic, sulfanilic,sulfuric, tartaric acid, and p-toluenesulfonic acid. Specific non-toxicacids include hydrochloric, hydrobromic, phosphoric, sulfuric, andmethanesulfonic acids. Examples of specific salts thus includehydrochloride and mesylate salts. Other examples of salts are well knownin the art, see, e.g., Remington's Pharmaceutical Sciences, 18th ed.,Mack Publishing, Easton Pa. (1990).

As used herein, the term “hydrate” means3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt thereof, that further includes astoichiometric or non-stoichiometric amount of water bound bynon-covalent intermolecular forces.

As used herein, the term “solvate” means3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt thereof, that further includes astoichiometric or non-stoichiometric amount of a solvent bound bynon-covalent intermolecular forces.

4.2 Diseases Associated with Premature Translation Termination

The invention encompasses methods of treating, preventing or managingdiseases or disorders ameliorated by the suppression of prematuretranslation termination and/or nonsense-mediated mRNA decay in a patientwhich comprise administering to a patient in need thereof an effectiveamount of an orally bioavailable compound (i.e.,3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof) accordingto the dosages and/or dosing regimens described herein.

In one embodiment, the present invention encompasses the treatment,prevention or management of any disease that is associated with a geneexhibiting premature translation termination and/or nonsense-mediatedmRNA decay. In one embodiment, the disease is due, in part, to the lackof expression of the gene resulting from a premature stop codon.Specific examples of genes which may exhibit premature translationtermination and/or nonsense-mediated mRNA decay and diseases associatedwith premature translation termination and/or nonsense-mediated mRNAdecay are found in U.S. Patent Application No. 60/390,747, titled:Methods For Identifying Small Molecules That Modulate PrematureTranslation Termination And Nonsense Mediated mRNA Decay, filed Jun. 21,2002, which is incorporated herein by reference in its entirety.

In a specific embodiment, the methods, compositions, doses, unit dosageforms and dosing regimens provided herein are useful for the treatment,prevention or management of a disease associated with a nonsensemutation in a gene in an embryo or fetus who has or is predisposed orsusceptible to a disease associated with a nonsense mutation in a gene,such as those described herein. In accordance with this embodiment, apregnant female is administered an effective amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof whichpasses through the placenta to the embryo or fetus. In a particularembodiment, an effective amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof isadministered orally to the pregnant female.

Diseases or disorders associated with or ameliorated by the suppressionof premature translation termination and/or nonsense-mediated mRNA decayinclude, but are not limited to: a genetic disease, cancer, anautoimmune disease, a blood disease, a collagen disease, diabetes, aneurodegenerative disease, a proliferative disease, a cardiovasculardisease, a pulmonary disease, an inflammatory disease or central nervoussystem disease.

Specific genetic diseases within the scope of the methods of theinvention include, but are not limited to, multiple endocrine neoplasia(type 1, 2 and 3), amyloidosis, mucopolysaccharidosis (type I and III),congenital adrenal hypoplasia, adenomatous poliposis coli, Von HippelLandau Disease, Menkes Syndrome, hemophilia A, hemophilia B, collagenVII, Alagille Syndrome, Townes-Brocks Syndrome, rhabdoid tumor,epidermolysis bullosa, Hurler's Syndrome, Coffin-Lowry Syndrome,aniridia, Charcot-Maria-Tooth Disease, myotubular myopathy, X-linkedmyotubular myopathy, X-linked chondrodysplasia, X-linkedagammaglobulinemia, polycystic kidney disease, spinal muscular atrophy,familial adenomatous poliposis, pyruvate dehydrogenase deficiency,phenylketonuria, neurofibromatosis 1, neurofibromatosis 2, Alzheimer'sdisease, Tay Sachs disease, Rett Syndrome, Hermansky-Pudlak Syndrome,ectodermal dysplasia/skin fragility syndrome, Leri-Weilldyschondrosteosis, rickets, hypophosphataemic, adrenoleukodystrophy,gyrate atrophy, atherosclerosis, sensorineural deafness, dystonia, DentDisease, acute intermittent porphyria, Cowden Disease, Herlitzepidermolysis bullosa, Wilson Disease, Treacher-Collins Syndrome,pyruvate kinase deficiency, giantism, dwarfism, hypothyroidism,hyperthyroidism, aging, obesity, Parkinson's disease, Niemann Pick'sdisease C, Cystic Fibrosis, muscular dystrophy, heart disease, kidneystones, ataxia-telangiectasia, familial hypercholesterolemia, retinitispigmentosa, lysosomal storage disease, tuberous sclerosis, DuchenneMuscular Dystrophy, and Marfan Syndrome.

In another embodiment, the genetic disease is an autoimmune disease. Ina preferred embodiment, the autoimmune disease is rheumatoid arthritisor graft versus host disease.

In another embodiment, the genetic disease is a blood disease. In apreferred embodiment, the blood disease is hemophilia A, Von Willebranddisease (type 3), ataxia-telangiectasia, b-thalassemia or kidney stones.

In another embodiment, the genetic disease is a collagen disease. In aembodiment, the collagen disease is osteogenesis imperfecta orcirrhosis.

In another embodiment, the genetic disease is diabetes.

In another embodiment, the genetic disease is an inflammatory disease.In a preferred embodiment, the inflammatory disease is arthritis.

In another embodiment, the genetic disease is a central nervous systemdisease. In one embodiment the central nervous system disease is aneurodegenerative disease. In a preferred embodiment, the centralnervous system disease is multiple sclerosis, muscular dystrophy,Duchenne muscular dystrophy, Alzheimer's disease, Tay Sachs disease,late infantile neuronal ceroid lipofuscinosis (LINCL) or Parkinson'sdisease.

In another embodiment, the genetic disease is cancer. In a preferredembodiment, the cancer is of the head and neck, eye, skin, mouth,throat, esophagus, chest, bone, lung, colon, sigmoid, rectum, stomach,prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine,heart or adrenals. The cancer can be primary or metastatic. Cancersinclude solid tumors, hematological cancers and other neoplasias.

In another preferred embodiment, the cancer is associated with tumorsuppressor genes (see e.g. Garinis et al. 2002, Hum Gen 111:115-117;Meyers et al. 1998, Proc. Natl. Acad. Sci. USA, 95: 15587-15591; Kung etal. 2000, Nature Medicine 6(12): 1335-1340. Such tumor suppressor genesinclude, but are not limited to, APC, ATM, BRAC1, BRAC2, MSH1, pTEN, Rb,CDKN2, NF1, NF2, WT1, and p53.

In a particularly preferred embodiment, the tumor suppressor gene is thep53 gene. Nonsense mutations have been identified in the p53 gene andhave been implicated in cancer. Several nonsense mutations in the p53gene have been identified (see, e.g., Masuda et al., 2000, Tokai J ExpClin Med. 25(2):69-77; Oh et al., 2000, Mol Cells 10(3):275-80; Li etal., 2000, Lab Invest. 80(4):493-9; Yang et al., 1999, Zhonghua ZhongLiu Za Zhi 21(2):114-8; Finkelstein et al., 1998, Mol Diagn. 3(1):37-41;Kajiyama et al., 1998, Dis Esophagus. 11(4):279-83; Kawamura et al.,1999, Leuk Res. 23(2):115-26; Radig et al., 1998, Hum Pathol.29(11):1310-6; Schuyer et al., 1998, Int J Cancer 76(3):299-303;Wang-Gohrke et al., 1998, Oncol Rep. 5(1):65-8; Fulop et al., 1998, JReprod Med. 43(2):119-27; Ninomiya et al., 1997, J Dermatol Sci.14(3):173-8; Hsieh et al., 1996, Cancer Lett. 100(1-2):107-13; Rall etal., 1996, Pancreas. 12(1):10-7; Fukutomi et al., 1995, Nippon Rinsho.53(11):2764-8; Frebourg et al., 1995, Am J Hum Genet. 56(3):608-15; Doveet al., 1995, Cancer Surv. 25:335-55; Adamson et al., 1995, Br JHaematol. 89(1):61-6; Grayson et al., 1994, Am J Pediatr Hematol Oncol.16(4):341-7; Lepelley et al., 1994, Leukemia. 8(8):1342-9; McIntyre etal., 1994, J Clin Oncol. 12(5):925-30; Horio et al., 1994, Oncogene.9(4):1231-5; Nakamura et al., 1992, Jpn J Cancer Res. 83(12):1293-8;Davidoff et al., 1992, Oncogene. 7(1):127-33; and Ishioka et al., 1991,Biochem Biophys Res Commun. 177(3):901-6; the disclosures of which arehereby incorporated by reference in their entireties).

In other embodiments, diseases to be treated, prevented or managed byadministering to a patient in need thereof an effective amount of a3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof include,but are not limited to, solid tumor, sarcoma, carcinomas, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, retinoblastoma, a blood-born tumor, acutelymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acutelymphoblastic T-cell leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute monoblastic leukemia, acuteerythroleukemic leukemia, acute megakaryoblastic leukemia, acutemyelomonocytic leukemia, acute nonlymphocyctic leukemia, acuteundifferentiated leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia, hairy cell leukemia, or multiple myeloma. Seee.g., Harrison's Principles of Internal Medicine, Eugene Braunwald etal., eds., pp. 491-762 (15th ed. 2001).

In other embodiments, diseases to be treated, prevented or managed byadministering to a patient in need thereof an effective amount of a3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof include,but are not limited to: retinitis pigmentosa (e.g., resulting from apremature stop codon in mRNA encoded by the ABCA4, PRP8, PRPF8, PRPF31,RPD1, RP1, RP2, RP6, RP9, RP10, RP14, RP17, RPGDB, RPGR or SRPX gene ora nonsense mutation in the ABCA4, PRP8, PRPF8, PRPF31, RPD1, RP1, RP2,RP6, RP9, RP10, RP14, RP17, RPGDB, RPGR or SRPX gene), neurofibromatosis(e.g., resulting from a premature stop codon in mRNA encoded by the NF1or NF2 gene or a nonsense mutation in the NF1 or NF2 gene),methylmalonic aciduria (e.g., due to methylmalonic CoA mutase deficiencyresulting from a premature stop codon in mRNA encoded by the MUT gene ora nonsense mutation in the MUT gene), Miyoshi myopathy (e.g., resultingfrom a premature stop codon in mRNA encoded by the dysferlin gene or anonsense mutation in the dysferlin gene), myotonic myopathy (e.g.,resulting from a premature stop codon in mRNA encoded by the HSPG2 orSJS1 gene or a nonsense mutation in the HSPG2 or SJS1 gene), limb-girdlemuscular dystrophy (e.g., resulting from a premature stop codon in mRNAencoded by the CAPN3, DYSF, LGMD, LGMD1A, LGMD1B, PLEC1, SGCB or SGCGgene or a nonsense mutation in the CAPN3, DYSF, LGMD, LGMD1A, LGMD1B,PLEC1, SGCB or SGCG gene), mucopolysaccharidosis type I (e.g., resultingfrom a premature stop codon in mRNA encoded by the IDUA gene or anonsense mutation in the IDUA gene), mucopolysaccharidosis type III A,mucopolysaccharidosis type IV, mucopolysaccharidosis type VI,mucopolysaccharidosis type VII or spinal muscular atrophy (e.g.,resulting from a premature stop codon in mRNA encoded by the SMA@, SMN1,SMN2, SMAD1, HMN2, SMAL or SMAX2 gene or a nonsense mutation in theSMA@, SMN1, SMN2, SMAD1, HMN2, SMAL or SMAX2 gene).

4.3 Doses and Dosing Regimens

Without being limited by theory, the present invention encompasses, inpart, specific doses and dosing regimens for3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof thatoptimize the suppression of premature translation termination and/ornonsense-mediated mRNA decay. In a preferred embodiment, thenonsense-mediated mRNA decay results from a nonsense mutation of DNA.

The novel methods of the invention encompass the treatment, preventionand management of diseases treatable or preventable by the suppressionof premature translation termination and/or nonsense-mediated mRNA decayor symptoms thereof while reducing or avoiding adverse or unwantedeffects, e.g., toxicities or side effects. The preferred route ofadministration for the doses and dosing regimens described herein isoral (i.e., ingestion of a solution, a colloid solution or a solutionwith additional active agent, above the saturating concentration ofactive agent).

The doses and dosing regimens described herein are thought to be usefuldue to their ability to achieve and maintain a desirable plasmaconcentration of the active agent. Without being limited by theory, itis thought that achieving and maintaining a relatively constant plasmaconcentration of active agent (such as those described in section 4.4)over, for example, a 24 hour period or longer, provides a beneficialtherapeutic effect to the patient. The doses and dosing regimensdescribed herein are useful for achieving and maintaining suchtherapeutic plasma concentrations of active agent.

In one embodiment, the invention relates to a method of administering3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof wherein theactive agent is administered to a patient in need thereof once in a 12or 24 hour period.

In another embodiment, the invention relates to a method ofadministering 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acidor a pharmaceutically acceptable salt, solvate or hydrate thereofwherein the active agent is administered to a patient in need thereoftwo times in a 12 or 24 hour period, wherein each administration ispreferably separated by about 4-14 hours, in one embodiment 12 hours. Inthese embodiments, the active agent can be administered, for example, atmeal time, such as breakfast and supper.

In another embodiment, the invention relates to a method ofadministering 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acidor a pharmaceutically acceptable salt, solvate or hydrate thereofwherein the active agent is administered to a patient in need thereofthree times in a 12 or 24 hour period, wherein each administration ispreferably separated by about 4-14 hours. In a particular embodiment,the active agent is administered once in the morning, once in theafternoon and once in the evening. Preferred intervals between dosesinclude 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 hours.

In one embodiment, the dose of active agent is escalated throughout a 24hour period. In another embodiment, the second dose administered isescalated (e.g., doubled). In another embodiment, the first and seconddose administered are kept constant and the third dose administered isescalated (e.g., doubled). In a particular embodiment, the three dosesin a 24 hour period are administered according to the formula: 1X, 1X,2X, where X is a particular initial dose (e.g., 4 mg/kg, 7 mg/kg or 10mg/kg). In another embodiment, the active agent is administered within(i.e., before or after) about 10, 15, 30, 45 or 60 minutes of thepatient having food. In one embodiment, an effective amount of theactive agent is sprinkled on or mixed in food. In another embodiment,the active agent is administered without food.

A particularly preferred dosing regimen is that where a patient isadministered the active agent within 30 minutes after a meal atapproximately 6-, 6-, and 12-hour intervals (e.g., at ˜7:00 AM afterbreakfast, ˜1:00 PM after lunch, and at ˜7:00 PM after supper).

In yet another embodiment, the invention relates to the administrationof 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof in singleor divided (e.g., three times in a 24 hour period) doses between 0.1mg/kg and 500 mg/kg, 1 mg/kg and 250 mg/kg, 1 mg/kg and 150 mg/kg, 1mg/kg and 100 mg/kg, 1 mg/kg and 50 mg/kg, 1 mg/kg and 25 mg/kg, 1 mg/kgand 10 mg/kg or 2 mg/kg and 10 mg/kg to a patent in need thereof. In aparticular embodiment,3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof isadministered in a dose of about 2-6 mg/kg, about 5-9 mg/kg, about 6-10mg/kg, about 8-12 mg/kg, about 12-16 mg/kg or about 18-22 mg/kg. In aparticular embodiment,3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof isadministered in a dose of about 4 mg/kg, about 7 mg/kg, about 8 mg/kg,about 10 mg/kg, about 14 mg/kg or about 20 mg/kg. In another embodiment,any dose of the 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid or a pharmaceutically acceptable salt, solvate or hydrate describedin the preceding embodiment is administered three times in a 24 hourperiod.

In another embodiment, the invention relates to continuous therapywherein 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof isadministered daily to a patient in need thereof for a certain period oftime (e.g., 5, 7, 10, 14, 20, 24, 28, 60 or 120 days or more). In oneembodiment, the active agent is continuously administered three timesper 24 hour period. In another embodiment, the active agent isadministered continuously daily, weekly, monthly or yearly. In aspecific embodiment, the active agent is continuously administered threetimes per 24 hour period at doses of about 4 mg/kg, about 4 mg/kg andabout 8 mg/kg for days, weeks, months or years. In a specificembodiment, the active agent is continuously administered three timesper 24 hour period at doses of about 7 mg/kg, about 7 mg/kg and about 14mg/kg for days, weeks, months or years. In a specific embodiment, theactive agent is continuously administered three times per 24 hour periodat doses of about 10 mg/kg, about 10 mg/kg and about 20 mg/kg for days,weeks, months or years. In each 24 hour period that the active agent isadministered, it is preferably administered three times at approximately6-, 6, and 12-hour intervals (e.g., at ˜7:00 AM after breakfast, ˜1:00PM after lunch, and at ˜7:00 PM after supper). Continuous therapy ispreferably used for the treatment, prevention or management of CysticFibrosis and Duchenne Muscular Dystrophy.

Treatment periods for a course of therapy can span one week, two weeks,three weeks, four weeks, five weeks, six weeks, seven weeks, eightweeks, nine weeks, ten weeks, eleven weeks, twelve weeks, thirteenweeks, fourteen weeks, four months, five months, six months, sevenmonths, eight months, nine months, ten months, eleven months, one year,two years, three years, four years, five years or longer. The treatmentperiods can be interrupted by periods of rest which can span a day, oneweek, two weeks, three weeks, four weeks, five weeks, six weeks, sevenweeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks,thirteen weeks, fourteen weeks, four months, five months, six months,seven months, eight months, nine months, ten months, eleven months, oneyear, two years, three years, four years, five years or longer. Suchdeterminations can be made by one skilled in the art (e.g., aphysician).

In a particular embodiment, treatment is continuous for 14 days,followed by no treatment for 14 days, followed by continuous treatmentfor an additional 14 days. In one embodiment, the dose given during thesecond 14 days of treatment is greater than that given during the first14 days of treatment. As a non-limiting example, a patient in needthereof is administered three doses of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof (e.g., 4mg/kg, 4 mg/kg and 8 mg/kg) in a 24 hour period for 14 continuous days,followed by 14 days without treatment, followed by administration ofthree doses of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid or a pharmaceutically acceptable salt, solvate or hydrate thereof(e.g., 10 mg/kg, 10 mg/kg and 20 mg/kg) in a 24 hour period for anadditional 14 continuous days.

In another embodiment, treatment is continuous for 28 days.

In certain embodiments,3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof isadministered according to the doses and dosing schedules describedherein in combination with a second active agent (e.g., simultaneouslyor sequentially). In particular embodiments,3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof isadministered according to the doses and dosing schedules describedherein in combination with an aminoglycoside, a corticosteroid, apancreatic enzyme, an antibiotic, insulin, a hypoglycemic agent, anomega-3 fatty acid, a chemotherapeutic agent, or an enzyme replacementtherapy. The administration of the second active agent can be topical,enteral (e.g. oral, duodenal, rectal), parenteral (e.g., intravenous,intraarterial, intramuscular, subcutaneous, intradermal orinteraperitoneal) or intrathecal. In certain embodiments,3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof isadministered according to the doses and dosing schedules describedherein in combination with radiation therapy.

It will be understood that the amounts of active agent administered to apatient in need thereof are or can be calculated based upon the actualweight of the patient in question or the average weight of the patientpopulation in question (e.g., white males, white females, AfricanAmerican males, African American females, Asian males or Asian females,including adults and children).

4.4 Plasma Concentrations

In one embodiment, the invention relates to a method of maintaining aplasma concentration of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof of greaterthan: about 0.1 μg/ml, about 0.5 μg/ml, about 2 μg/ml, about 5 μg/ml,about 10 μg/ml, about 20 μg/ml, about 25 μg/ml, about 40 μg/ml, about 50μg/ml, about 100 μg/ml, about 150 μg/ml, about 200 μg/ml, about 250μg/ml or about 500 μg/ml in a patient for at least about 2, 2.5, 3, 3.5,4, 4.5, 5, 6, 8, 12 or 24 hours or longer, comprising administering aneffective amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof to apatient in need thereof. In a particular embodiment, the administrationis oral. Levels of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid or a pharmaceutically acceptable salt, solvate or hydrate thereofin plasma can be measured, for example, by high performance liquidchromatography (HPLC).

In another embodiment, the invention relates to a method of maintaininga plasma concentration of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof of about0.1 μg/ml to about 500 μg/ml or about 2 μg/ml to about 10 μg/ml in apatient for at least about 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 12 or 24hours or longer, comprising administering an effective amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof to apatient in need thereof one, two or three times per day at the same orescalating doses (e.g., 1X, 1X, 2X as described herein). In a particularembodiment, the administration is oral.

In a particular embodiment, a patient's plasma level of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof ismaintained above about 2 μg/ml for at least about 2, 2.5, 3, 3.5, 4,4.5, 5, 6, 8, 12 or 24 hours or longer by administration of the activeagent one, two or three times per day to a patient in need thereof. Inanother embodiment, a patient's plasma level of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof ismaintained between about 2 μg/ml to about 10 μg/ml for at least about 2,2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 12 or 24 hours or longer hours byadministration of the active agent one, two or three times per day to apatient in need thereof. In a particular embodiment, a patient's plasmalevel of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof ismaintained above about 10 μg/ml for at least about 2, 2.5, 3, 3.5, 4,4.5, 5, 6, 8, 12 or 24 hours or longer by administration of the activeagent one, two or three times per day to a patient in need thereof. In aparticular embodiment, the administration is oral.

4.5 Patient Populations

Particular patient populations which the methods and compositions of thepresent invention are useful for include adults and children who have orare susceptible to having (e.g., due to environmental or geneticfactors) a disease associated with a nonsense mutation, such as thosedescribed herein.

In one embodiment, it has been determined through pre-screening that thepatient or a relative of the patient has a nonsense mutation (i.e., UAA,UGA, or UAG).

4.6 Pharmaceutical Compositions and Unit Dosage Formulations

Pharmaceutical compositions and single unit dosage forms comprising3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof are alsoencompassed by the invention. Individual dosage forms of the inventionmay be suitable for oral, mucosal (including sublingual, buccal, rectal,nasal, or vaginal) or parenteral (including subcutaneous, intramuscular,bolus injection, intraarterial, or intravenous) administration.Preferred pharmaceutical compositions and single unit dosage forms aresuitable for oral administration.

In one embodiment, the pharmaceutical composition is a solid oral dosageform. In one embodiment, the pharmaceutical composition is a liquid oraldosage form. In a particular embodiment, present invention providesdoses, unit dosage formulations and pharmaceutical compositions wherein3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof is orallybioavailable. Advantages of oral administration can include ease ofadministration, higher patient compliance with the dosing regimen,clinical efficacy, fewer complications, shorter hospital stays, andoverall cost savings.

In another embodiment, the invention relates to unit dosage formulationsthat comprise between about 35 mg and about 1400 mg, about 125 mg andabout 1000 mg, about 250 mg and about 1000 mg, or about 500 mg and about1000 mg of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid ora pharmaceutically acceptable salt, solvate or hydrate thereof. In oneembodiment, the unit dosage formulation comprises3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof and one ormore carriers or excipients suitable for suspension in apharmaceutically acceptable solvent (e.g., water, milk, a carbonatedbeverage, juice, apple sauce, baby food or baby formula) in a bottle.

In another embodiment, the invention relates to unit dosage formulationsthat comprise 35 mg, 50 mg, 70 mg, 100 mg, 125 mg, 140 mg, 175 mg, 200mg, 250 mg, 280 mg, 350 mg, 500 mg, 560 mg, 700 mg, 750 mg, 1000 mg or1400 mg of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid ora pharmaceutically acceptable salt, solvate or hydrate thereof.Preferred unit dosage formulations comprise about 125 mg, about 250 orabout 1000 mg of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid or a pharmaceutically acceptable salt, solvate or hydrate thereof.In one embodiment, the unit dosage formulation comprises3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof and one ormore carriers or excipients suitable for suspension in apharmaceutically acceptable solvent (e.g., water, milk, a carbonatedbeverage, juice, apple sauce, baby food or baby formula) in a bottle.Preferred unit dosage formulations are powders and sachets.

While it is recommended that the unit dosage formulations describedherein are stored at between about 2° C. to about 8° C., the unit dosageformulations can be stored at room temperature for about 48 hours priorto reconstitution. In one embodiment, reconstitution of a 250 mg unitdosage formulation of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof is carriedout by the addition of about 10 mL of water directly in a bottlecontaining 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid ora pharmaceutically acceptable salt, solvate or hydrate thereof toachieve a concentration of about 25 mg/mL in the total volume ofsuspension. For a 1000 mg unit dosage formulation of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof, about 20mL of water is added directly in the bottle containing3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof to achievea concentration of about 50 mg/mL in the total volume of suspension.Immediately after water is added, the bottle is capped and shaken gentlyby hand for at least about 30 seconds to achieve a homogeneoussuspension. Although the reconstituted suspension may remain in theoriginal plastic bottle for up to 24 hours before ingestion, it isrecommended that the drug be taken shortly after reconstitution. Ifthere is a delay of more than about 15 minutes between reconstitutionand dosing, it is recommended that the bottle should be reshaken gentlyby hand for at least about 30 seconds. It is recommended that thesuspension be administered directly from the bottle. If the entire unitdosage form is to be administered, it is further recommended that thebottle be rinsed once with water and this rinse water be ingested toensure that no powder is left in the bottle. If a partial amount of theunit dosage form is to be administered, a spoon or syringe can be usedto obtain the appropriate dose.

Single unit dosage forms of the invention suitable for oraladministration to a patient include, but are not limited to: sachets;cachets; tablets; caplets; capsules, such as soft elastic gelatincapsules; troches; lozenges; dispersions; powders; solutions; liquiddosage forms, including suspensions (e.g., aqueous or non-aqueous liquidsuspensions); emulsions (e.g., oil-in-water emulsions, or a water-in-oilliquid emulsion); and elixirs. In one embodiment, the invention relatesto a colloid solution or a solution with additional active agent, abovethe saturating concentration. These and other ways in which specificdosage forms encompassed by this invention will vary from one anotherwill be readily apparent to those skilled in the art. See, e.g.,Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, EastonPa. (1990).

This invention further encompasses anhydrous pharmaceutical compositionsand dosage forms comprising3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof. Anhydrouspharmaceutical compositions and dosage forms of the invention can beprepared using anhydrous or low moisture containing ingredients and lowmoisture or low humidity conditions.

Typical oral dosage forms of the invention are prepared by combining theactive ingredient(s) in an intimate admixture with at least one carrieror excipient according to conventional pharmaceutical compoundingtechniques. Excipients can take a wide variety of forms depending on theform of preparation desired for administration. For example, excipientssuitable for use in oral liquid or aerosol dosage forms include, but arenot limited to, water, glycols, oils, alcohols, flavoring agents (e.g.,vanilla extract), preservatives, and coloring agents. Examples ofexcipients suitable for use in solid oral dosage forms (e.g., powders,tablets, sachets, capsules, and caplets) include, but are not limitedto, starches, sugars, micro-crystalline cellulose, diluents, granulatingagents, lubricants, binders, and disintegrating agents.

Particularly preferred unit dosage formulations are powder formulationscomprising an effective amount of the active agent which are suitablefor reconstitution in a pharmaceutically acceptable solvent (e.g.,water, milk, a carbonated beverage, juice, apple sauce, baby food orbaby formula) and subsequent oral administration. In a particularembodiment, the powder can optionally contain one or more carriers orexcipients in combination with the active agent. In another embodiment,the powder can be stored in a sealed container prior to administrationor reconstitution. In yet another embodiment, the powder can beencapsulated (e.g., in a gelatin capsule).

Liquid preparations for oral administration may take the form of, forexample, solutions, syrups or suspensions, or they may be presented as adry product (e.g., powder or granule) for constitution with water orother suitable vehicle before use. Such liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, cellulosederivatives or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, flavoring, coloring andsweetening agents as appropriate.

Examples of excipients that can be used in solid oral dosage forms ofthe invention include, but are not limited to, binders, fillers,disintegrants, and lubricants. Binders suitable for use inpharmaceutical compositions and dosage forms include, but are notlimited to, corn starch, potato starch, or other starches, gelatin,natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropylmethyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystallinecellulose, and mixtures thereof.

Preferred excipients include Litesse® Ultra (refined polydextrose)mannitol, surfactant agents (polyethylene glycol 3350 and Lutrol® microF127 (poloxamer 407 powder)), a disintegrant (crospovidone), cab-o-sil,Carbopol®, polyacrylic acid and other excipients (hydroxyethylcellulose, vanilla flavor, magnesium stearate (non-bovine), andcolloidal silica).

Examples of fillers suitable for use in the pharmaceutical compositionsand solid dosage forms disclosed herein include, but are not limited to,lactose, talc, calcium carbonate (e.g., granules or powder),microcrystalline cellulose, powdered cellulose, dextrates, kaolin,mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, andmixtures thereof. The binder or filler in pharmaceutical compositions ofthe invention is typically present in from about 50 to about 99 weightpercent of the pharmaceutical composition or dosage form.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Aspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

5. EXAMPLES

The following examples are offered by way of illustration and notlimitation.

5.1 Example 1 Preparation of3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid

To a solution of 3-Cyanobenzoic acid (44.14 g, 300 mmol) in DMF (0.6 L)was added K₂CO₃ (62.19 g, 450 mmol) and then stirred for 30 min at roomtemperature. To the suspension was added methyl iodide (28 mL, 450 mmol)over 20 min, and the reaction mixture was stirred further 4 h at roomtemperature. The reaction mixture was poured to 1.2 L of ice water andstirred for 30 min, and the precipitate was filtered off. The white cakewas dissolved in methanol (70 mL), and then re-precipitated in coldwater. The desired product was obtained as a white powder with 79% yield(38 g, 99% purity by LC/UV). ¹H-NMR (CDCl₃) δ 8.85 (2H), 8.28 (1H), 8.02(1H), 4.17 (3H).

To a solution of 3-Cyanobenzoic acid methyl ester (50 g, 310 mmol) inethanol (500 mL) was added 50% aqueous hydroxylamine (41 mL, 620 mmol)at room temperature. The reaction mixture was stirred for 1 h at 100° C.and the solvents were removed under reduced pressure. The oily residuewas dissolved in 20/80 ethanol/toluene (50 mL×2) and then concentratedagain. The desired ester (61 g, quan. yield) was obtained as a whitepowder with 98% purity (LC/UV). ¹H-NMR (CDCl₃) δ 9.76 (1H), 8.24 (1H),7.82 (2H), 7.51 (1H), 5.92 (2H), 3.82 (3H).

To a solution of 3-(N-Hydroxycarbamimidoyl)-benzoic acid methyl ester(60 g, 310 mmol) in anhydrous THF (200 mL) was addeddiisopropylethylamine (75 mL, 434 mmol) at 5° C., and then to themixture was added 2-fluorobenzoyl chloride (48.1 mL, 403 mmol) over 20min. The reaction mixture was stirred for 1 h at room temperature. Theprecipitate was filtered off and the filtrate was concentrated underreduced pressure. The residue was dissolved in ethylacetate (400 mL) andthen washed with water (200 mL×2). The solvent was removed under reducedpressure and the desired product was crystallized in 60% ethylacetate inhexane to yield the desired product (81 g, 83% yield) as a white solid.¹H-NMR (CDCl₃) δ 8.18 (1H), 8.03 (3H), 7.48 (2H), 7.18 (2H), 5.61 (2H),3.82 (3H).

44 g of 3-(N-2-Fluorobenzoylcarbamimidoyl)-benzoic acid methyl ester intoluene (500 mL) was refluxed for 4 h at 130° C. using Dean-Starkapparatus. The reaction mixture was stirred at 5° C. for 18 h. The whiteprecipitate was filtered off and the filtrate was concentrated,crystallized again in toluene. The desired oxadiazole (38 g, 92% yield)was obtained as a white solid with 99% purity (LC/UV). ¹H-NMR (CDCl₃) δ8.91 (1H), 8.38 (1H), 8.15 (2H), 7.62 (2H), 7.35 (2H), 3.95 (3H).

To a solution of 3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid methyl ester (3.3 g, 11 mmol) in THF (40 mL) was added 1.5M aqueousNaOH (10 mL, 14 mmol). The reaction mixture was refluxed for 2 h at 100°C. The organic solvent was removed and the aqueous solution was dilutedwith water (50 mL), and then acidified with aqueous HCl. The whiteprecipitate was filtered off and the white cake was washed with coldwater and then dried using lyophilizer. The desired acid (3.0 g, 96%yield) was obtained as a white powder with 98% purity (LC/UV). Meltingpoint 242° C.; IR 3000 (Aromatic C—H), 1710 (C═O); ¹H-NMR (D₆-DMSO) δ8.31 (1H), 8.18 (2H), 8.08 (1H), 7.88 (2H), 7.51 (2H); ¹³C-NMR (D₆-DMSO)δ 172.71, 167.38, 166.48, 161.25, 135.80, 132.24, 131.79, 131.79,131.08, 130.91, 129.81, 127.76, 125.48, 117.38, 111.70; ¹⁹F-NMR(D₆-DMSO) δ 109.7.

Pharmaceutically acceptable salts of3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid can beprepared using methods known to those skilled in the art. The sodiumsalt can be prepared as follows. To a solution of3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid methyl ester(33 g, 111 mol) in THF (400 mL) was added 1.5M aqueous NaOH (100 mL, 144mmol). The reaction mixture was refluxed for 2 h at 100° C. The organicsolvent was removed under reduced pressure and the aqueous solution wasstirred 2 h at 5° C. The white precipitate was filtered off and thefiltrate was concentrated and precipitated again in water. The whitecake was washed with cold water and then dried using lyophilizer. Thedesired salt (33 g, 96% yield) was obtained as a white powder with 98.6%purity (LC/UV).

5.2 Example 2 Oral Treatment of Nonsense-Mutation-Mediated CysticFibrosis

The present example sets forth an illustrative dosing regimen useful forthe treatment of nonsense-mutation-mediated Cystic Fibrosis.

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof is providedas a vanilla-flavored powder for suspension. The drug is manufacturedunder current Good Manufacturing Practice conditions (cGMP). Theformulation can include binding and suspending agents, surfactants, andvarious minor excipients that aid in the manufacturing process. Themixture can be packaged in 40 mL plastic (high-density polyethylene[HDPE]) bottles sealed with a foil seal and a white plastic, childproofcap. Each bottle can contain 125, 250 or 1000 mg of the drug substance,which is 25.0% of the total formulation weight. Alternatively, themixture can be provided in a sachet formulation, such as set forth inExample 6. Excipients (and their proportions of the total formulationweight) include a suspending agent (Litesse® Ultra [refinedpolydextrose]—25.7%), a binding agent that can also providetaste-masking (mannitol—25.0%), surfactant agents (polyethylene glycol3350—12.8% and Lutrol® micro F127 [poloxamer 407 powder]—3.7%), adisintegrant (crospovidone—5.0%), and other excipients, each less than2% (hydroxyethyl cellulose, vanilla flavor, magnesium stearate[non-bovine], and colloidal silica) can be present. Bottle labelsindicate the identity of the drug substance, the lot number, the amountof the drug substance, and the storage conditions (e.g., roomtemperature or refrigeration at 5° to 8° C.).

Dosing of the drug substance is based on milligrams of drug per kilogramof patient body weight. The dose of the drug substance can be rounded tobe consistent with the available bottle sizes. The dosing scheme ensuresthat the total actual dose given is never <50 mg below or >250 mg abovethe intended dose (i.e., is always within 5 mg/kg of the assigned doselevel). For example, a patient weighing 40 kg being treated with the 4mg/kg dose would have a calculated dose of 160 mg. This patient wouldreceive one 250 mg bottle (250 mg total) or 6.25 mg/kg/dose. The samepatient when treated with the 8 mg/kg dose in the evening would have acalculated dose of 320 mg and would receive two 250 mg bottles (500 mgtotal) or 12.5 mg/kg. The same patient treated with the 10 mg/kg dosewould have a calculated dose of 400 mg and would receive two 250 mgbottles (500 mg total) or 12.5 mg/kg. The same patient when treated withthe 20 mg/kg dose in the evening would have a calculated dose of 800 mgand would receive one 1000 mg bottle (1000 mg total) or 25 mg/kg.

The reconstitution and dosing of the drug product is done at roomtemperature. No specific warming of the drug product is necessary beforereconstitution. The drug product can be reconstituted with anypharmaceutically acceptable solvent (e.g., water, milk, a carbonatedbeverage, juice, apple sauce, baby food or baby formula). For each 250mg bottle provided, ˜10 mL of water or other pharmaceutically acceptablesolvent is added to achieve a concentration of about 25 mg/mL in thetotal volume of suspension. For each 1000 mg bottle provided, ˜20 mL ofwater or other pharmaceutically acceptable solvent is added to achieve aconcentration of about 50 mg/mL in the total volume of suspension.Immediately after water or other pharmaceutically acceptable solvent isadded to the dry study medication, the bottle(s) is capped and shakenvigorously by hand for about 60 seconds to achieve homogeneity ofsuspension. Although the suspension may remain in the original plasticbottle for up to 24 hours before ingestion, it is recommended that thedrug be taken shortly after reconstitution. If there is a delay of morethan 15 minutes between reconstitution and dosing, the bottle should bereshaken vigorously by hand for about 60 seconds.

Treatment is administered continuously for as long as necessary to apatient having or susceptible to having Cystic Fibrosis. Table 1 setsforth illustrative daily dosing regimens for3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof whereinadministration occurs three times per day at 6-, 6-, and 12-hourintervals (e.g., ˜7:00 AM, ˜1:00 PM and ˜7:00 PM) with food. In aparticular embodiment, the patient is administered3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof as setforth in Table 1 continuously for 14 days, followed by 14 days withouttreatment, followed by an additional 14 days of administration, followedby an additional 14 days without treatment. In another particularembodiment, the patient is administered3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof as setforth in Table 1 continuously for 14 days at three daily doses of 4mg/kg, 4 mg/kg and 8 mg/kg, followed by 14 days without treatment,followed by an additional 14 days of administration at three daily dosesof 10 mg/kg, 10 mg/kg and 20 mg/kg, followed by an additional 14 dayswithout treatment. In certain embodiments, a single daily dosing regimenset forth in Table 1 is followed each day. In other embodiments,different dosing regimens set forth in Table 1 can be followed ondifferent days.

TABLE 1 Dosing Scheme 1 2 3 Regimen TID dosing TID dosing TID dosingwith food with food with food Schedule Continuous Continuous ContinuousDaily Admin. Daily Admin. Daily Admin. Time Dose ~7:00 AM 4 mg/kg  7mg/kg 10 mg/kg ~1:00 PM 4 mg/kg  7 mg/kg 10 mg/kg ~7:00 PM 8 mg/kg 14mg/kg 20 mg/kg Abbreviations: TID = three times per day

Patients preferably take the drug within 30 minutes after a meal;ideally the drug will be taken at approximately 6-, 6, and 12-hourintervals (e.g., at ˜7:00 AM after breakfast, ˜1:00 PM after lunch, andat ˜7:00 PM after supper). Patients ingest the drug by filling eachbottle with the required amount of water or other pharmaceuticallyacceptable solvent, capping and shaking each bottle for about 60seconds, and then ingesting the contents of the required number and sizeof bottles per dose. The entire dose of reconstituted drug is to betaken at one time. After ingestion, each dosing bottle is half-filledwith water or another pharmaceutically acceptable solvent, capped andshaken, and this water or other pharmaceutically acceptable solvent fromthe bottle is ingested by the patient. This rinse procedure is carriedout once. In certain embodiments, the drug is provided as a sachet. Inthese embodiments, the appropriate amount of the drug can be weighed ormeasured and combined with an appropriate pharmaceutically acceptablesolvent prior to administration.

5.3 Example 3 Oral Treatment of Nonsense-Mutation-Mediated DuchenneMuscular Dystrophy

The present example sets forth an illustrative dosing regimen useful forthe treatment of nonsense-mutation-mediated Duchenne Muscular Dystrophy.

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof is providedas a vanilla-flavored powder for suspension. The drug is manufacturedunder current Good Manufacturing Practice conditions (cGMP). Theformulation can include binding and suspending agents, surfactants, andvarious minor excipients that aid in the manufacturing process. Themixture can be packaged in 40 mL plastic (high-density polyethylene[HDPE]) bottles sealed with a foil seal and a white plastic, childproofcap. Each bottle can contain 125, 250 or 1000 mg of the drug substance,which is 25.0% of the total formulation weight. Alternatively, themixture can be provided in a sachet formulation, such as set forth inExample 6. Excipients (and their proportions of the total formulationweight) include a suspending agent (Litesse® Ultra [refinedpolydextrose]—25.7%), a binding agent that can also providetaste-masking (mannitol—25.0%), surfactant agents (polyethylene glycol3350—12.8% and Lutrol® micro F127 [poloxamer 407 powder]—3.7%), adisintegrant (crospovidone—5.0%), and other excipients, each less than2% (hydroxyethyl cellulose, vanilla flavor, magnesium stearate[non-bovine], and colloidal silica) can be present. Bottle labelsindicate the identity of the drug substance, the lot number, the amountof the drug substance, and the storage conditions (e.g., roomtemperature or refrigeration at 5° to 8° C.).

Dosing of the drug is based on milligrams of drug per kilogram ofpatient body weight. The total volume corresponding to the totalmilligram amount of drug to be administered to a patient should becalculated. For example, if a 30-kg patient is to get 4 mg/kg, then thedose to be delivered will be 30×4=120 mg. This patient should be dosedusing the 250 mg dose bottle. Since each mL of the suspension in the 250mg dose bottle contains 250/10=25 mg of the drug, this patient shouldget 120/25=˜5 mL of the suspension for each 4 mg/kg dose). The samepatient when treated with the 8 mg/kg dose in the evening would have acalculated dose of 240 mg and would receive one 250 mg bottle (10 mLsuspension). These volumes of the suspensions for the respective dosesshould be withdrawn from the drug bottle using a plastic oral dosingsyringe. For transfer of fractional volumes of <10 (for 250 mg bottle)or <20 mL (for 1000 mg bottle), the desired amount should be withdrawnfrom the study medication bottle into a dosing syringe of an appropriatetype and size (e.g., a Baxa, Exacta-Med, calibrated, latex-free,plastic, oral dosing syringe) and dosed using the same syringe. Duringthe same 24 hours after reconstitution, >1 dose may be taken from thesame bottle of suspension; however, reconstituted drug should not bestored beyond 24 hours with the intention of using this material againfor multiple doses in the same patient. If the total amount of drug tobe taken in 1 day exceeds 10 mL (for 250 mg bottle) or 20 mL (for 1000mg bottle) of the reconstituted drug, then a new bottle of drug shouldbe used for each dosing.

The reconstitution and dosing of the drug product is done at roomtemperature. No specific warming of the drug product is necessary beforereconstitution. The drug can be reconstituted with any pharmaceuticallyacceptable solvent (e.g., water, milk, a carbonated beverage, juice,apple sauce, baby food or baby formula). For each 250 mg bottleprovided, ˜10 mL of water or other pharmaceutically acceptable solventis added to achieve a concentration of about 25 mg/mL in the totalvolume of suspension. For each 1000 mg bottle provided, ˜20 mL of wateror other pharmaceutically acceptable solvent is added to achieve aconcentration of about 50 mg/mL in the total volume of suspension.Immediately after water or other pharmaceutically acceptable solvent isadded to the dry study medication, the bottle(s) is capped and shakenvigorously by hand for about 60 seconds to achieve homogeneity ofsuspension. Although the suspension may remain in the original plasticbottle for up to 24 hours before ingestion, it is recommended that thedrug be taken shortly after reconstitution. If there is a delay of morethan 15 minutes between reconstitution and dosing, the bottle should bereshaken vigorously by hand for about 60 seconds.

Treatment is administered continuously for as long as necessary to apatient having or susceptible to having Duchenne Muscular Dystrophy.Table 2 sets forth illustrative daily dosing regimens for3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof whereinadministration occurs three times per day at 6-, 6-, and 12-hourintervals (e.g., ˜7:00 AM, ˜1:00 PM and ˜7:00 PM) with food. In aparticular embodiment, the patient is administered3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof in one ofthe dosing regimens set forth in Table 2 continuously for 28 days. Incertain embodiments, a single daily dosing regimen set forth in Table 2is followed each day. In other embodiments, different dosing regimensset forth in Table 2 can be followed on different days. In certainembodiments, the drug is provided as a sachet. In these embodiments, theappropriate amount of the drug can be weighed or measured and combinedwith an appropriate pharmaceutically acceptable solvent prior toadministration.

TABLE 2 Dosing Scheme 1 2 3 Regimen TID dosing TID dosing TID dosingwith food with food with food Schedule Continuous Continuous ContinuousDaily Admin. Daily Admin. Daily Admin. Time Dose ~7:00 AM 4 mg/kg  7mg/kg 10 mg/kg ~1:00 PM 4 mg/kg  7 mg/kg 10 mg/kg ~7:00 PM 8 mg/kg 14mg/kg 20 mg/kg Abbreviations: TID = three times per day

Patients are administered the drug within 30 minutes after a meal;ideally the drug will be taken at approximately 6-, 6, and 12-hourintervals (e.g., at ˜7:00 AM after breakfast, ˜1:00 PM after lunch, andat ˜7:00 PM after supper). Patients ingest the drug by filling eachbottle with the required amount of water or other pharmaceuticallyacceptable solvent, capping and shaking each bottle for about 60seconds, withdrawing the appropriate amount of volume from the bottleusing an oral dosing syringe and ingesting the contents directly fromthe dosing syringe. The entire calculated volume of reconstituted drugcorresponding to the dose is to be taken at one time. After ingestion ofthe drug, the dosing syringe should be filled with the same volume ofwater or other pharmaceutically acceptable solvent as the dose volume,and should be ingested by the patient. This rinse procedure should becarried out once.

Efficacy of treatment can be determined by measuring the change from abaseline measurement of dystrophin levels in a biopsy of the foot muscleextensor digitorum brevis (EDB).

5.4 Example 4 Preparation of Unflavored Dosages of3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or aPharmaceutically Acceptable Salt, Solvate or Hydrate Thereof

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof is providedas a powder for suspension. The drug is manufactured under current GoodManufacturing Practice conditions (cGMP). The drug can be intimatelymixed with binding and suspending agents, surfactants, and various minorexcipients that aid in the manufacturing process. The mixture ispackaged in a 40 mL plastic (high-density polyethylene [HDPE]) bottlesealed with a foil seal and a white plastic, childproof cap. Each bottlecan contain about 35 mg, about 70 mg, about 125 mg, about 140 mg, about175 mg, about 250 mg, about 280 mg, about 350 mg, about 560 mg, about700 mg, about 1000 mg or about 1400 mg of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof. Excipients(and their proportions of the total formulation weight) optionallyinclude a suspending agent (Litesse® Ultra [refinedpolydextrose]—25.7%), a binding agent that can also providetaste-masking (mannitol—25.0%), surfactant agents (polyethylene glycol3350—12.8% and Lutrol® micro F127 [poloxamer 407 powder]—3.7%), adisintegrant (crospovidone—5.0%), and other excipients, each less than2% (cab-o-sil, hydroxyethyl cellulose, magnesium stearate [non-bovine],and colloidal silica) can be present. The bottle is then labeled toindicate the identity of the drug substance, the lot number, the amountof the drug substance, and the storage conditions (e.g., refrigerationat 5° to 8° C.). Prior to administration, the drug product isreconstituted in an appropriate volume of a pharmaceutically acceptablesolvent (e.g., water, milk, a carbonated beverage, juice, apple sauce,baby food or baby formula).

5.5 Example 5 Preparation of Flavored Dosages of3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or aPharmaceutically Acceptable Salt, Solvate or Hydrate Thereof

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof is providedas a vanilla-flavored (e.g., by addition of vanilla extract) powder forsuspension. The drug is manufactured under current Good ManufacturingPractice conditions (cGMP). The drug can be intimately mixed withbinding and suspending agents, surfactants, and various minor excipientsthat aid in the manufacturing process. The mixture is packaged in a 40mL plastic (high-density polyethylene [HDPE]) bottle sealed with a foilseal and a white plastic, childproof cap. Each bottle can contain about35 mg, about 70 mg, about 125 mg, about 140 mg, about 175 mg, about 250mg, about 280 mg, about 350 mg, about 560 mg, about 700 mg, about 1000mg or about 1400 mg of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof. Excipients(and their proportions of the total formulation weight) optionallyinclude a suspending agent (Litesse® Ultra [refinedpolydextrose]—25.7%), a binding agent that can also providetaste-masking (mannitol—25.0%), surfactant agents (polyethylene glycol3350—12.8% and Lutrol® micro F127 [poloxamer 407 powder]—3.7%), adisintegrant (crospovidone—5.0%), and other excipients, each less than2% (cab-o-sil, hydroxyethyl cellulose, vanilla flavor, magnesiumstearate [non-bovine], and colloidal silica) can be present. The bottleis then labeled to indicate the identity of the drug substance, the lotnumber, the amount of the drug substance, and the storage conditions(e.g., refrigeration at 2° to 8° C.). Prior to administration, the drugproduct is reconstituted in an appropriate volume of a pharmaceuticallyacceptable solvent (e.g., water, milk, a carbonated beverage, juice,apple sauce, baby food or baby formula). The drug product can be storedat room temperature for up to 48 hours prior to reconstitution.

A number of references have been cited, the entire disclosure of whichare incorporated herein by reference in their entirety.

5.6 Example 6 Sachet Formulation of3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or aPharmaceutically Acceptable Salt, Solvate or Hydrate Thereof

The mixture is packaged using a pouch or sachet that is comprised ofmultiple laminated layers that may include a paper layer, an aluminumfoil layer and a surlyn layer. Each sachet can contain about 125 mg,about 250 mg, about 500 mg or about 1000 mg of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof. Excipients(and their proportions of the total formulation weight) optionallyinclude either of the following as set forth in Table 3 and Table 4.

TABLE 3 Formulation Ingredient Weight %3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3- 25.0 yl]-benzole acid or apharmaceutically acceptable salt, solvate or hydrate thereof Litesse ®Ultra 24.75 Polyethylene Glycol 12.8 Lutrol ® Micro 3.7 Mannitol 25.0Hydroxyethyl Cellulose 1.5 Vanilla Flavor 0.75 Crospovidone 5.0Cab-o-sil 0.5 Magnesium Stearate 0.5 Talc 0.5

TABLE 4 Formulation Ingredient Weight %3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3- 25.0 yl]-benzoic acid or apharmaceutically acceptable salt, solvate or hydrate thereof Litesse ®Ultra 25.65 Polyethylene Glycol 12.8 Lutrol ® Micro 3.7 Mannitol 25.0Hydroxyethyl Cellulose 1.5 Vanilla Flavor 0.75 Crospovidone 5.0Cab-o-sil 0.1 Magnesium Stearate 0.5

The sachet is then labeled to indicate the identity of the drugsubstance, the lot number, the amount of the drug substance, and thestorage conditions (e.g., refrigeration at 2° to 8° C.). Prior toadministration, an appropriate amount of the drug product isreconstituted in an appropriate volume of a pharmaceutically acceptablesolvent (e.g., water, milk, a carbonated beverage, juice, apple sauce,baby food or baby formula). The drug product can be stored at roomtemperature for up to 48 hours prior to reconstitution.

5.7 Example 7 Transepithelial Potential Difference (TEPD) Assay

The measurement of transepithelial potential difference (TEPD), alsoknown as nasal potential difference, provides a sensitive evaluation ofsodium and chloride transport directly in secretory epithelial cells viaassessment of transepithelial bioelectric properties (Knowles et al.,1981, N. Engl. J. Med. 305(25):1489-95; Knowles et al., 1995, Hum. GeneTher. 6:445). TEPD is performed in each nostril using standardizedtechniques (Standaert et al., 2004, Ped. Pulm. 37:385-92). In theprocedure, a small plastic catheter is used to assess electricaldifferences across the outer cell membrane of nasal mucosa cells in thenostril. TEPD values are expressed in millivolts, or mV. A chlorideconductance equal to or more electrically negative than −5.0 mV isgenerally considered to be in the normal range. TEPD assessments aremade on the nasal epithelium cells lining the inferior turbinate becausethese cells are easier to access than the respiratory epithelial cellslining the lower airways, and have been shown to have the same iontransport characteristics (Knowles et al., 1981, Am. Rev. Respir. Dis.124(4):484-90). TEPD assessments can also be made on rectal epithelialcells and lower respiratory epithelial cells. Because of the role of theCFTR protein in transporting chloride ions across cell membranes, andbecause of the absence of this protein, cystic fibrosis patients have anabnormal TEPD chloride conductance. As an endpoint, TEPD has theadvantage that it can detect chloride transport changes that are aquantitative integration of the presence, functional activity, andapical location of the CFTR in airway cells. Furthermore, it is a directmeasure of CFTR activity that is not likely to be affected by supportiveor palliative treatments for CF (with the possible exception ofsystemically administered aminoglycoside antibiotics). Of importance isevidence that TEPD values can correlate with the degree of pulmonarydysfunction and radiographic abnormality (Ho et al., 1997, Eur. Respir.J. 10(9):2018-22; Fajac et al., 1998, Eur. Respir. J. 12(6):1295-300;Sermet-Gaudelus et al., 2005, Am. J. Respit. Crit. Care Med.171(9):1026-1031). In particular, TEPD assessment ofisoproterenol-induced CFTR chloride activity has demonstrated betterpredictive value than genotype in determining FEV1 and radiologicalscore (Ho et al., 1997, Eur Respir J. 10(9):2018-22). Under baselineconditions, TEPD-assessed chloride channel activity is very unlikely tonormalize spontaneously in patients with CF; any observed improvementsin TEPD-assessed chloride channel activity are expected to specificallydenote pharmacological activity of CFTR-correcting therapies.Accordingly, it has become the primary endpoint in Phase 1-2pharmacological and gene replacement studies aimed at correcting CFTRdysfunction (Peckham et al., 1995, AJ. Clin Sci (London). 89(3):277-84;Wilschanski et al., 2003, N. Engl. J. Med. 349(15):1433-41).

5.8 Example 8 CFTR Immunofluorescence

The collection and processing of the nasal mucosal curettage from eachnostril of a patient for measurement of CFTR protein byimmunofluorescence and by quantification of CFTR mRNA is performed usingstandardized techniques (Clancy et al., 2001, Am. J. Respir. Crit. CareMed. 163(7):1683-92; Amaral et al., 2004, J. Cyst. Fibros. 3 Suppl2:17-23). The immunofluorescence staining of normal epithelial cells(for example, from nasal mucosal scrapings) reveals the presence of mostof the CFTR protein at the apical surface. In animal models ofnonsense-mutation-mediated CF or in patients withnonsense-mutation-mediated CF, CFTR staining is absent (e.g., inpatients homozygous for a premature stop mutation) or is primarilyobserved in the perinuclear region (e.g., in patients with a ΔF508mutation that prevents normal CFTR intracellular trafficking).Successful production of functional wild or non-wild type CFTR proteinin both animal models and patients has been associated with reappearanceof apical epithelial CFTR protein as assessed by immunofluorescence(Clancy et al., 2001, Am. J. Respir. Crit. Care Med. 163(7):1683-92;Wilschanski et al., 2003, N. Engl. J. Med. 349(15):1433-41).

5.9 Example 9 Pulmonary Function Tests

Pulmonary function tests, including FEV₁, FVC, and MEF₂₅₋₇₅, aremeasured using standard spirometry procedures. Assessments of pulmonaryfunction (including MEF₂₅₋₇₅, FVC, and, particularly, FEV₁) have beenacknowledged as definitive clinical endpoints in patients with CF (Foodand Drug Administration, 62_(nd) Anti-Infective Drugs AdvisoryCommittee. Discussion of NDA for tobramycin solution for inhalation(Tobi®) for the management of cystic fibrosis patients. November, 1997;Tiddens, 2002, Pediatr. Pulmonol. 34(3): 228-31). FEV₁ and otherpulmonary function testing measures have been shown to correlate withdisease severity, predict morbidity in terms of health care utilizationand IV antibiotic usage, and indicate the risk of CF-related mortality(Food and Drug Administration, 62_(nd) Anti-Infective Drugs AdvisoryCommittee. Discussion of NDA for tobramycin solution for inhalation(Tobi®) for the management of cystic fibrosis patients. November, 1997).Pulmonary function testing is simple to administer (even in patients asyoung as 7 years of age), and uses standardized equipment and techniquesthat are widely available. Interpretation is performed usingwell-established normative equations that account for patient age,height, and gender. Improvement in FEV₁ has been acknowledged asquantitatively demonstrating meaningful clinical benefit in CF, and hasserved as the basis for regulatory approval of dornase alfa and inhaledtobramycin (Food and Drug Administration, 62_(nd) Anti-Infective DrugsAdvisory Committee. Discussion of NDA for tobramycin solution forinhalation (Tobi®) for the management of cystic fibrosis patients.November, 1997).

5.10 Example 10 Phase 2 Study of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic as an OralTreatment for Nonsense-Mutation-Mediated Cystic Fibrosis

Patients must have met all of the following conditions to be eligiblefor enrollment into the study:

1. Diagnosis of CF based on documented evidence of a conclusivelyabnormal sweat test (sweat chloride >60 mEq/liter by pilocarpineiontophoresis (LeGrys, Sweat testing: Sample collection and quantitativeanalysis: Approved guidelines—Second edition. National Committee forClinical Laboratory Standards 2000; Vol 20:14));

2. Abnormal chloride secretion as measured by TEPD (a more positive than−5 mV TEPD assessment of chloride secretion with chloride-free amilorideand isoproterenol);

3. Presence of a nonsense mutation in one of the alleles of the cftrgene;

4. Documentation that cftr gene sequencing has been performed;

5. Age ≧18 years;

6. Body weight ≧40 kg;

7. FEV₁≧40% of predicted for age, gender, and height (Knudson standards)(Knudson, 1983, Am. Rev. Respir. Dis. 127: 725-734);

8. Oxygen saturation (as measured by pulse oximetry)≧92% on room air;

9. Willingness of male and female patients, if not surgically sterile,to abstain from sexual intercourse or employ a barrier or medical methodof contraception during the study drug administration and follow-upperiods;

10. Negative pregnancy test (for females of childbearing potential);

11. Willingness and ability to comply with scheduled visits, drugadministration plan, study procedures (including TEPD measurements,clinical laboratory tests, and PK sampling), and study restrictions;

12. Ability to provide written informed consent; and

13. Evidence of personally signed and dated informed consent documentindicating that the patient has been informed of all pertinent aspectsof the trial.

The presence of any of the following conditions excluded a patient fromenrollment in the study:

1. Prior or ongoing medical condition (e.g., concomitant illness,psychiatric condition, alcoholism, drug abuse), medical history,physical findings, ECG findings, or laboratory abnormality that, in theinvestigator's opinion, could adversely affect the safety of thepatient, makes it unlikely that the course of treatment or follow-upwould be completed, or could impair the assessment of study results;

2. Ongoing acute illness including acute upper or lower respiratoryinfections within 2 weeks before start of study treatment;

3. History of major complications of lung disease (including recentmassive hemoptysis or pneumothorax) within 2 months prior to start ofstudy treatment;

4. Abnormalities on screening chest x-ray suggesting clinicallysignificant active pulmonary disease other than CF, or new, significantabnormalities such as atelectasis or pleural effusion which may beindicative of clinically significant active pulmonary involvementsecondary to CF;

5. Positive hepatitis B surface antigen, hepatitis C antibody test, orhuman immunodeficiency virus (HIV) test;

6. Hemoglobin <10 g/dL;

7. Serum albumin <2.5 g/dL;

8. Abnormal liver function (serum total bilirubin>the upper limit ofnormal, or serum ALT, AST, or GGT >2.0 times the upper limit of normal);

9. Abnormal renal function (serum creatinine >1.5 times upper limit ofnormal);

10. Pregnancy or breast-feeding;

11. History of solid organ or hematological transplantation;

12. Exposure to another investigational drug within 14 days prior tostart of study treatment;

13. Ongoing participation in any other therapeutic clinical trial;

14. Ongoing use of thiazolidinedione peroxisome proliferator-activatedreceptor gamm (PPAR γ) agonists, eg, rosiglitazone (Avandia® orequivalent) or pioglitazone (Actos® or equivalent);

15. Change in intranasal medications (including use of corticosteroids,cromolyn, ipratropium bromide, phenylephrine, or oxymetazoline) within14 days prior to start of study treatment;

16. Change in treatment with systemic or inhaled corticosteroids within14 days prior to start of study treatment;

17. Use of or requirement for inhaled gentamicin or amikacin within 14days prior to start of study treatment or during study treatment; or

18. Requirement for systemic aminoglycoside antibiotics within 14 daysprior to start of study treatment.

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid was providedin a formulation described herein. 15 patients (12 from a Phase 2 trialbeing conducted in Israel and 3 from a Phase 2 trial being conducted inthe United States; seven patients were male and 8 were female; patientshad a median age of 22 years; and all patients had multiple signs andsymptoms of cystic fibrosis, including some degree of lung dysfunction)were orally administered3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid according tothe following 56 day schedule: administration of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid three timesper day (TID) at 4 mg/kg, 4 mg/kg and 8 mg/kg for 14 days, followed byno treatment for 14 days (Cycle 1, consisting of 28 days), followed byadministration of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoicacid three times per day (TID) at 10 mg/kg, 10 mg/kg and 20 mg/kg for 14days, followed by no treatment for 14 days (Cycle 2, consisting of 28days).

Clinical endpoints were evaluated using the procedures set forth above.TEPD measurements were made prior to treatment and on days 14 and 28 ofCycle 1 and Cycle 2. Nasal mucosal curettage was collected from eachnostril of each patient prior to treatment and on days 14 and 28 ofCycle 1 and Cycle 2. Pulmonary tests, including FEV₁, FVC and MEF₂₅₋₇₅,were measured prior to treatment, on day −1 of Cycle 2, on day 13 or 14of Cycle 1 and day 13 or 14 of Cycle 2 in the study being conducted inIsrael and the same parameters were measured prior to treatment and onday 13 or 14 of Cycle 2 in the study being conducted in the UnitedStates.

Mean change in TEPD chloride conductance. This is the average of thechanges from the beginning to the end of the treatment period in TEPDchloride conductance within each study participant. For example, if thechanges in TEPD chloride conductance within each of three participantswere −7.0 mV, −2.0 mV and −9.0 mV, the mean change in TEPD chlorideconductance among these participants would be −6.0 mV.

Percentage of patients with a chloride conductance response. This is thepercentage of patients who demonstrated a TEPD chloride conductanceresponse at the end of treatment with3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid. For purposesof the trials, a chloride conductance response is defined as a TEPDchloride conductance improvement of at least −5 mV. For example, in apatient with a TEPD chloride conductance value of +1.0 mV at baselineand a TEPD chloride conductance value of −6.0 mV at the end oftreatment, the TEPD chloride conductance improvement would be −7.0 mV,representing a chloride conductance response.

Percentage of patients with improvements of TEPD chloride conductancevalues into the normal range. As noted above, a chloride conductanceequal to or more electrically negative than −5.0 mV is generallyconsidered to be in the normal range. As such, a patient with a TEPDchloride conductance value of +1.0 mV at baseline would be considered tohave an abnormal value because the value is more electrically positivethan −5.0 mV. If, at the end of treatment, that patient's TEPD chlorideconductance value improved to −6.0 mV, this would represent animprovement into the normal range because the improved value is moreelectrically negative than −5.0 mV.

Based on patient gender, age and height, the mean FEV₁ value at studyentry was 66% of normal and the mean FVC value at study entry was 80% ofnormal. Fourteen of the 15 patients included in the analysis had airwaycolonization with Pseudomonas aeruginosa, a common bacterial infectionin cystic fibrosis patients that can lead to serious pneumonia. Fourteenof the 15 patients also had pancreatic insufficiency and requiredchronic pancreatic enzyme replacement therapy. Patients had low bodyweights, with a mean weight of 58.3 kg at study entry.

Table 5 presents the TEPD results for the 5 patients. For eachmeasurement, the results are presented on a best-of-nostrils andmean-of-both-nostrils basis. Historically, results of TEPD tests havetypically been presented on a best-of-nostrils basis. However, recentguidelines established by the Cystic Fibrosis Therapeutics DevelopmentNetwork recommend that TEPD results be presented on both bases.Improvements in TEPD chloride conductance in patients with differenttypes of nonsense mutations within the CFTR gene were noted.

TABLE 5 Lower Dose Level Higher Dose Level TEPD Result Result p-ValueResult p-Value Mean change in TEPD chloride conductance: Best ofnostrils −9.0 mV <0.001 −6.4 mV 0.010 Mean of both −6.7 mV <0.001 −4.4mV 0.023 nostrils Number of patients with ≧ −5 mV improvement in TEPDchloride conductance: Best of nostrils 9/15 (60%) <0.001 8/15 (53%)<0.001 Mean of both 6/15 (40%) 0.005 7/15 (47%) <0.001 nostrils Numberof patients with improvement in TEPD chloride conductance to normal:Best of nostrils 8/15 (53%) 0.008 8/15 (53%) 0.008 Mean of both 6/15(40%) 0.032 7/15 (47%) 0.016 nostrils

The treatment effects at the lower and the higher3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid dose levelswere not statistically significant, suggesting that further doseescalation may not be necessary and that even lower doses of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid may beeffective in improving TEPD chloride conductance. Statisticallysignificant results and positive trends for secondary endpoints werealso observed. In particular, although the trials were not been poweredto detect statistical significant changes in secondary endpoints,statistically significant improvements from study entry to the end ofthe higher-dose treatment cycle in the patients' mean FEV₁, FVC andweight were observed. Table 6 presents the results. For the changes inlung function, one patient was not included because that patient did nothave lung function measured at the end of the higher-dose treatmentcycle.

TABLE 6 Endpoint End of Lung function (expressed Higher as a percentageof normal Study Dose for gender, age and height): Entry Treatment Changep-Value Mean FEV₁ 65.8% 69.1% 3.3% 0.015 Mean FVC 80.2% 85.1% 4.9% 0.037Weight 58.3 kg 59.0 kg 0.7 kg 0.012

In addition, although changes in patient's symptoms were not formallymeasured through the use of a quality-of-life questionnaire, trialinvestigators were requested to ask about changes in patients' cysticfibrosis symptoms. In the 15 patients included in the interim analysis,6 reported general improvements in well being, 6 reported decrease incough and 10 reported decreased mucus thickness and easier clearing ofmucus.

5.11 Example 11 Dystrophin, Sarcoglycan, and Dystroglycan Expression byImmunofluorescence and Western Blotting

Biopsy of the EDB muscle and overlying skin from one foot is performedunder local anesthesia and conscious sedation (in some cases, generalanesthesia may be required) prior to treatment, and from the other footon the last day of treatment. The biopsy procedure is performed usingstandardized techniques (Stedman, 2000, Human Gene Therapy 11:777-90).The entire muscle belly (whenever possible) is removed in the procedure.At the time of collection of the biopsy prior to treatment, the musclespecimen is divided into at least 3 fragments and the biopsy specimencollected on the last day of treatment is divided into at least 2fragments. The biopsy specimen is placed on a telfa gauze spongemoistened with Ringer's saline. The biopsy specimen is viewed at lowpower under a stereo dissection microscope to establish fiberorientation. The muscle is then transected using a sharp scalpel in across sectional fashion (perpendicular to the orientation of the fibers)whenever possible and allowed to rest for 2 minutes to allow for thecessation of spasm. The sample is then frozen in liquid nitrogen cooledisopentane, transferred to a liquid nitrogen reservoir and held 1 inchabove the liquid/vapor interface for 2 minutes of slow cooling andisopentane evaporation before immersion in the liquid nitrogen, andwrapped into precooled (in liquid nitrogen and stored on dry ice) foillabeled with the study number, site number, patient number, date,patient initials, and foot side (right foot or left foot).

All sample containers are clearly labeled in a fashion that identifiesthe subject and the collection date. Labels are fixed to the samplecontainers in a manner that prevents the label from becoming detached.Samples are shipped for analysis/culture/central review immediatelyafter the procedure is performed. For detection of dystrophin, 3commercially available antibodies that recognize the C-terminus, theN-terminus, and the rod domain of the protein are employed. Fordetection of the sarcoglycan and dystroglycan complex, commerciallyavailable antibodies against α-, β-, γ-, and δ-sarcoglycan, andβ-dystroglycan are used when possible. Epifluorescence microscopy isused in the analysis; images are captured by CCD camera, afternormalization of the fluorescence intensity against a normal musclespecimen. Images are stored digitally and preserved for future review,and final evaluation at the completion of the study. Tissues are alsoprocessed for detection of dystrophin, the sarcoglycans, andβ-dystroglycan by Western blotting using the same antibodies.Microscopic images are captured and preserved for future review, and forfinal evaluation at the completion of the study. Remaining muscle tissuesamples are preserved for confirmatory assays of mRNA and proteinsinvolved in DMD. Immunostaining and Western blotting are employed forprotein detection.

Muscle biopsies are commonly performed on DMD subjects as a component ofdiagnosis and as measures of therapeutic effect in the context ofresearch studies. EDB has been chosen because it is not an essentialmuscle for daily activities and therefore sampling this muscle does nothave adverse functional consequence for the subject. Because it islittle used, the EDB muscle is unlikely to demonstrate substantialfibrotic replacement of muscle and thus provides an appropriate tissuefor detection of dystrophin production. Sampling of the EDB muscleoffers additional practical advantages because it is easy to identify,can be dissected under local anesthesia, and provides sufficient amountsof tissue to carry out the required analyses. Immunofluorescence andWestern blotting are routine tests performed on muscle biopsy specimensto confirm the presence or absence of full-length dystrophin. An absenceof dystrophin is viewed as confirmation of the diagnosis of DMD.Restoration of dystrophin, with localization to the muscle membrane, hasbeen considered a direct measure of preclinical and clinicalpharmacodynamic activity (Barton-Davis, 1999, J. Clin. Invest.104(4):375-81; Politano, 2003, Acta Myol. 22(1):15-21).

5.12 Example 12 Upper and Lower Extremity Myometry

Upper and lower extremity myometry are performed using a hand-heldmyometer following standardized procedures (Beenakker, 2001,Neuromuscul. Disord. 11(5):441-6; Hyde, 2001, Neuromuscul. Disord.11(2):165-70). It is recommended (depending on the subject's baselinefunctional status) that evaluated muscle groups include hip abductors,knee extensors, elbow flexors and extensors, and hand grip. Bilateralassessments can be done, and three measurements can be recorded fromeach muscle group on each side. These parameters are monitored prior totreatment, on the second to last day of treatment, and during afollow-up period after treatment. During the pre-treatment and treatmentperiods, the myometry procedures are performed prior to the musclebiopsy.

Myometry assessments using a hand-held dynamometer are a sensitive andreproducible measure of muscle strength in ambulatory and non-ambulatorysubjects (Beenakker, 2001, Neuromuscul. Disord. 11(5):441-6; Hyde, 2001,Neuromuscul. Disord. 11(2):165-70). Inter-rater reliability in subjectswith muscular dystrophy is high (Stuberg, 1988, Phys. Ther. 198868(6):977-82; Hyde, 2001, Neuromuscul. Disord. 11(2):165-70). Ascompared to manual muscle strength testing, myometry is a more sensitiveand less complex measure of muscle function (McDonald, 1995, Am. J.Phys. Med. Rehabil. (5 Suppl):S70-92). The test can be readilyadministered by the evaluator (e.g., physician or physical therapist).

5.13 Example 13 Timed Function Tests

Timed function tests include time taken to stand from a supine position,time taken to walk 10 meters, and time taken to climb 4 standard-sizedstairs (Mendell, 1989, N. Engl. J. Med. 320(24):1592-7; Griggs, 1991,Arch. Neurol. 48(4):383-8). These parameters are monitored prior totreatment, on the second to last day of treatment, and during afollow-up period after treatment. During the pre-treatment and treatmentperiods, the timed function tests are performed prior to the musclebiopsy.

These tests (time taken to stand from supine position, time taken towalk 10 meters, and time taken to climb 4 standard-sized steps) providean additional measure of functional capability in ambulatory subjects.The tests are reproducible, commonly employed, simple to administer, andhave documented response to therapeutic intervention with steroids(Mendell, 1989, N. Engl. J. Med. 320(24):1592-7; Griggs, 1991, Arch.Neurol. 48(4):383-8).

5.14 Example 14 Serum CK Levels

Serum CK activity is assessed using a commercially available NADH-linkedkinetic assay (Diagnostic Chemicals Ltd., Oxford, Conn.). Serum CKlevels are measured prior to treatment, on day 1 (prior to first dose),day 7, day 14, day 21, and day 27 during the treatment period, and onday 42 and day 56 after treatment. Serum CK is increased in Duchennemuscular dystrophy and therefore is a readily measurable diagnosticmarker for the disease and may serve as a potential biomarker for thepharmacological activity of the drug (Mendell et al., 1989, New Eng. J.Med. 320(24):1592-1597).

Serum CK provides a measure of whole-body muscle integrity.Concentrations of this enzyme in the serum are increased 50- to 100-foldin subjects with DMD and measurements of its levels are used in makingan early diagnosis of the disease (Worton, The muscular dystrophies, In:Scriver C. R., Beaudet A. L., Sly W. S., Valle D, eds. The metabolic andmolecular basis of inherited disease. 8th ed. Vol. 4. New York:McGraw-Hill, 2001:5493-523). The levels of serum CK are measured tomonitor the progression of the disease and serve as a marker for muscledamage. While exercise-induced changes introduce variability (Politano,2003, Acta. Myol. 22(1):15-21), the marker has advantages because it canbe easily, repeatedly, and frequently assessed with a widely availableand reliable assay. Prior clinical studies have shown decreases in serumCK coincident with improvements in muscle strength during treatment withsteroids (Reitter, 1995, Brain Dev. 17 Suppl:39-43).

5.15 Example 15 Dermal Fibroblast and Muscle Cell Culture

Studies are performed on muscle tissue and skin from patients todetermine whether dystrophin production in primary muscle cultures fromthe patients corresponds with dystrophin production in vivo. Theseexperiments evaluate whether dermal fibroblasts from patients, whendifferentiated into muscle cells in vitro by transfection with aMyo-D-producing expression construct (Wang, 2001, Development 128:4623-33), demonstrate dystrophin production in response to treatment.Correlations of skin cell response with clinical activity may offer aneasy-to-obtain predictive test in selecting future patients for therapyor for screening new agents for the treatment of DMD. Cells are culturedas follows. Biopsy material is stored during transport in humanproliferation medium (or PBS), and on ice for longer time periods ifnecessary. If the tissue is not prepared within 24 hours, the materialcan be frozen in human proliferation medium containing 10% DMSO andstored in liquid nitrogen (or dry ice). At the time the tissue is to beprepared for setting up the myoblast culture, biopsy material is washedin PBS. PBS sufficient to keep the tissue moist is added into a culturedish. The biopsy material is minced thoroughly with razor blades, towardan almost homogeneous suspension. Approximately 2 ml ofcollegenase/dispase/CaCl₂ solution per gram of tissue is added andmincing is continued for several minutes (e.g. for a muscle biopsy of5×5×5 mm use 1 ml of enzyme solution). The suspension is transferredinto a sterile tube and incubated at 37° C. in a waterbath until themixture is a fine slurry (e.g., about 20 to 30 minutes). The suspensionis further homogenized by pipetting up and down several times duringincubation. Additional resuspension cycles by pipetting up an down witha syringe can be performed if necessary. Eight mL of human proliferationmedium is added to the suspension and mixed. The mixture is centrifugedfor 10 minutes at 1200 rpm. The cell pellet is resuspended in 3 ml humanproliferation medium. Cells are plated into one well of acollagen-coated 6-wells plate, or, depending on the amount of material,in a T25 collagen-coated flask. Cells are cultured for 48 hrs, at 37° C.and 5% CO₂. Non-attached cells are removed and transferred to anothercollagen-coated well (as backup). Fresh proliferation medium is added tothe first well (3 ml). The cells are cultured from the first well toconfluency and until two confluent T75-flasks have been obtained. Forstorage, cells can be frozed from one T75 flask into 4 cryotubes with 1ml freezing medium. The myogenic cell content of the culture isdetermined by performing a desmin-staining Preplating of the cultures isrequired if the percentage of desmin-positive cells is too low.

5.16 Example 16 Phase 2 Study of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic as an OralTreatment for Duchenne Muscular Dystrophy

Subjects must meet all of the following conditions to be eligible forenrollment into the study:

1. Diagnosis of Duchenne muscular dystrophy (DMD) based on a clinicalphenotype presenting by age 5, with increased serum CK and absence ofdystrophin on a muscle biopsy (negative sarcolemmal staining with anantibody to the C-terminal portion of the dystrophin protein);

2. Presence of a nonsense mutation in the dystrophin gene;

3. Documentation that dystrophin gene sequencing has been performed or,if sequencing has not already been performed, that a blood sample hasbeen sent for the confirmatory dystrophin gene sequencing;

4. Physical examination or radiographic imaging evidence of EDB musclesin both feet;

5. Ability to ambulate;

6. Male sex;

7. Age ≧5 years;

8. Willingness to abstain from sexual intercourse or employ a barrier ormedical method of contraception during the study drug administration andfollow-up periods in subjects known to be sexually active;

9. Willingness and ability to comply with scheduled visits, drugadministration plan, laboratory tests, study restrictions, and studyprocedures (including muscle biopsies, myometry, and PK sampling);

10. Able to provide written informed consent if ≧18 years of age, orwritten informed assent (with parental/guardian consent) if ≧7 years ofage. If the subject is <7 years of age, parent/legal guardian consentalone will be obtained; and

11. Evidence of personally signed and dated informed consent document(assent also required for children ≧7 years of age) indicating that thesubject/parent/legal guardian has been informed of all pertinent aspectsof the trial should be followed.

The presence of any of the following conditions will exclude a subjectfrom study enrollment:

1. Prior or ongoing medical condition (e.g., concomitant illness,psychiatric condition, alcoholism, drug abuse), medical history,physical findings, ECG findings, or laboratory abnormality that, in theinvestigator's opinion, could adversely affect the safety of thesubject, makes it unlikely that the course of treatment or follow-upwould be completed, or could impair the assessment of study results;

2. Clinical symptoms and signs of congestive cardiac failure (AmericanCollege of Cardiology/American Heart Association Stage C or Stage D)(Hunt, 2001, J. Am. Coll. Cardiol. 38:2101-13);

3. Positive hepatitis B surface antigen, hepatitis C antibody test, orhuman immunodeficiency virus (HIV) test;

4. Hemoglobin <10 g/dL;

5. Serum albumin <2.5 g/dL;

6. Abnormal GGT or total bilirubin (>laboratory's upper limit ofnormal);

7. Abnormal renal function (serum creatinine >1.5 times laboratory'supper limit of normal);

8. History of solid organ or hematological transplantation;

9. Ongoing immunosuppressive therapy (other than corticosteroids);

10. Exposure to another investigational drug within 28 days prior tostart of study treatment;

11. Ongoing participation in any other therapeutic clinical trial;

12. Ongoing use of thiazolidinedione peroxisome proliferator-activatedreceptor gamma (PPAR γ) agonists, e.g., rosiglitazone (Avandia® orequivalent) or pioglitazone (Actos® or equivalent);

13. Change in systemic corticosteroid therapy (e.g., initiation oftreatment; cessation of treatment; change in dose, schedule, or type ofsteroid) within 3 months prior to start of study treatment; or

14. Treatment with systemic aminoglycoside antibiotics within 3 monthsprior to start of study treatment.

3-[5-(2-Fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid is providedin a formulation described herein. Treatment is administered over 28days for each treatment cohort. An initial cohort of patients aretreated daily for 28 days with3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid at a givendose level (e.g., 4-, 4-, and 8-mg/kg) TID. If the initial patientstolerate the drug, then a second cohort of patients receives3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid at a higherdose level (e.g., 10-, 10-, and 20-mg/kg) TID. Thus, each patientreceives a total of 84 doses of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid. After theend of 28 days of treatment, each patient is followed for an additional28 days without treatment.

At each dose level, it is recommended that3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid be taken TIDat 6-, 6-, and 12-hour (±˜30 minutes) intervals. Ideally each dose istaken within ˜30 minutes after a meal (e.g., ˜7:00 AM after breakfast,˜1:00 PM after lunch, and ˜7:00 PM after dinner). While it is realizedthat variations in dosing schedule may occur in the outpatient setting,it is recommended that the prescribed regimen (including dosingintervals and the relationship of dosing to meals) be followed closelyon the days of PK sample collection. Clinical endpoints are evaluatedusing the procedures set forth above.

It will be appreciated that, although specific embodiments of theinvention have been described herein for purposes of illustration, theinvention described herein is not to be limited in scope by the specificembodiments herein disclosed. These embodiments are intended asillustrations of several aspects of the invention. Any equivalentembodiments are intended to be within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description.

What is claimed is:
 1. A method for treating a patient having CysticFibrosis or Duchenne Muscular Dystrophy associated with a lack ofexpression of a gene resulting from a premature stop codon or a nonsensemutation, comprising administering to the patient an oral pharmaceuticalcomposition comprising 25.0% by weight of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt thereof; 25.7% by weight of refinedpolydextrose; 25.0% by weight of mannitol; 12.8% by weight of asurfactant selected from either or both polyethylene glycol 3350 and3.7% by weight of poloxamer 407 powder; 5.0% by weight of crospovidone;and less than 2% by weight of one or more excipients selected fromhydroxyethyl cellulose, vanilla flavor, non-bovine magnesium stearate orcolloidal silica, wherein the method comprises administration of threedoses of 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt thereof in a 24 hour period accordingto the formula: 1X, 1X, 2X, and wherein said administration maintains aplasma concentration of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt thereof in a range of from about 2μg/mL to about 20 μg/mL in said patient for the 24 hour period.
 2. Themethod of claim 1, wherein said oral pharmaceutical compositioncomprises from 35 mg to 1400 mg of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt thereof in a unit dosage formulation.3. The method of claim 2, wherein the amount of3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid or apharmaceutically acceptable salt thereof in the unit dosage formulationis selected from 35 mg, 50 mg, 70 mg, 100 mg, 125 mg, 140 mg, 175 mg,200 mg, 250 mg, 280 mg, 350 mg, 500 mg, 560 mg, 700 mg, 750 mg, 1000 mg,or 1400 mg.
 4. The method of claim 1, wherein the oral pharmaceuticalcomposition is a unit dosage formulation packaged in plastic bottles. 5.The use of claim 1, wherein the oral pharmaceutical composition is apowder or granules packaged in a sachet.
 6. The method of claim 1,wherein the oral pharmaceutical composition is reconstituted as asuspension to achieve a concentration selected from 25 mg/mL in thetotal volume or 50 mg/mL in the total volume.
 7. The method of claim 1,wherein the oral pharmaceutical composition is reconstituted usingwater, milk, a carbonated beverage, juice, apple sauce, baby food orbaby formula.
 8. The method of claim 1, wherein the gene is the CFTRgene.
 9. The method of claim 1, wherein the gene is the dystrophin gene.10. The method of claim 1, wherein the three doses are administeredorally to a human.
 11. The method of claim 1, wherein the first dose isadministered in the morning, the second dose is administered at middayand the third dose is administered in the evening of the 24 hour period.12. The method of claim 1, wherein the second dose is administered about6 hours after the first dose is administered, wherein the third dose isadministered about 6 hours after the second dose is administered andwherein the first dose is administered about 12 hours after the thirddose was administered in a preceding 24 hour period, and wherein thefirst dose, the second dose, or the third dose are administered about 30minutes after a meal.
 13. The method of claim 1, wherein the three dailydoses are administered in a continuous therapy comprising a plurality of24 hour periods.
 14. The method of claim 1, wherein X is about 8 mg/kgto about 12 mg/kg.
 15. The method of claim 1, wherein X is about 10mg/kg.